Protein prenylation is a critically important post-translational modification process. It is required for the proper membrane association and activity of many signal transduction proteins, including the Ras oncogene products. There has been intense interest in the development of protein farnesyltransferase (FTase) inhibitors as anti-cancer agents, and promising results have been observed with these compounds in clinical trials. However, it is now clear that Ras is not the sole, or even the most important target of FTase inhibitors. The long-term goal of this research project is the development of chemical tools for the selective modulation of protein prenylation. These probes will allow for the determination of the relative roles of various prenylated proteins in cancer cell growth. This proposal will address the following specific hypothesis: The unique structure and mechanism of FTase allows for the discovery of selective inhibitors that will block the prenylation of certain proteins but not interfere with the prenylation of others. We will use currently existing and newly synthesized chemical tools, in combination with new biochemical and biological collaborative studies, to address the following specific aims: Aim 1) To characterize the interplay of peptide and isoprenoid selectivity in FTase and GGTase I. These studies will use existing and newly synthesized isoprenoid analogs, concise targeted synthetic peptide libraries, and various bioanalytical methods. Rational design will be employed along with screening methods to establish effective selectivity patterns. Aim 2) To elucidate the events covering selective prenylation by FTase at a molecular level, using existing kinetic techniques. Detailed steady state and pre-steady state kinetic experiments, in combination with various binding constant determinations, will be employed. These studies will provide novel insight into the mechanism employed by FTase, and insight into the design of new selective FPP analogs. Aim 3) To establish the ability of isoprenoid analogs to alter FTase substrate selectivity in cells, through the direct quantitation of prenyl isoforms of Ras and other prenylated proteins. The effects of farnesol analogs on flux through the mevalonate pathway and intracellularprenyl diphosphate pools also will be determined. The accomplishment of these aims will provide the tools and lay the groundwork for future studies to investigate the long-term hypothesis of this project: the selective introduction of modified prenyl groups into proteins will alter their subcellular localization and biological activity.